Effect of linker and spacer on the design of a fibronectin-mimetic peptide evaluated via cell studies and AFM adhesion forces

The design of a fibronectin-mimetic peptide that specifically binds to the alpha 5beta 1 integrin has been widely studied because of this integrin's participation in many physiological and pathological processes. A promising design for such a peptide includes both the primary binding site RGD and the synergy site PHSRN connected by a linker and extended off of a surface by a spacer. Our original hypothesis was that the degree of hydrophobicity/hydrophilicity between the two sequences (RGD and PHSRN) in fibronectin is an important parameter in designing a fibronectin-mimetic peptide (Mardilovich, A.; Kokkoli, E. Biomacromolecules 2004, 5, 950-957). A peptide-amphiphile, PR_b, that was previously designed in our laboratory employed a hydrophobic tail connected to the N terminus of a peptide headgroup that was composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. Even though our previous work (Mardilovich, A.; Craig, J. A.; McCammon, M. Q.; Garg, A.; Kokkoli, E. Langmuir 2006, 22, 3259-3264) demonstrated that PR_b is a promising sequence compared to fibronectin, this is the first study that tests our hypothesis by comparing PR_b to other peptides with hydrophobic or hydrophilic linkers. Furthermore, different peptide-amphiphiles were designed that could be used to study the effect of building blocks systematically, such as the peptide headgroup linker length and hydrophobicity/hydrophilicity as well as the headgroup spacer length on integrin adhesion. Circular dichroism spectroscopy was first employed, and the collected spectra demonstrated that only one peptide-amphiphile exhibited a secondary structure. Their surface topography was evaluated by taking atomic force microscopy (AFM) images of Langmuir-Blodgett peptide-amphiphile membranes supported on mica. Their adhesion was first evaluated with AFM force measurements between the different sequences and an AFM tip functionalized with purified integrins. The amphiphiles were further characterized via 1-12 h cell studies that examined human umbilical vein endothelial cell adhesion and extracellular matrix fibronectin production. The AFM studies were in good agreement with the cell studies. Overall, the adhesion studies validated our hypothesis and demonstrated for the first time that a "neutral" linker, which more closely mimics the cell adhesion domain of fibronectin, supports higher levels of adhesion compared to other peptide designs with a hydrophobic or hydrophilic linker or even fibronectin. Neutral linker lengths that were within the distance found between PHSRN and RGD in fibronectin performed equally well. However, the 10 amino acid neutral linker gave slightly better cell adhesion than did the control fibronectin at all times. Also, a short spacer was shown to give higher adhesion than other sequences with no spacer or a longer spacer, suggesting that a short spacer is necessary to extend the sequence further away from the interface. In conclusion, this work outlines a logical approach that can be applied for the rational design of any protein-mimetic peptide with two binding sites.     

Design of a novel fibronectin-mimetic peptide-amphiphile for functionalized biomaterials

The interaction of the alpha5beta1 integrin with its ligand, fibronectin, supports numerous adhesive functions and has an important role in health and disease. In recent years, there has been a considerable effort in designing fibronectin-mimetic peptides to target the integrin. However, to date, the therapeutic use of these peptides has been limited, as they cannot accurately mimic fibronectin's binding affinity for alpha5beta1. A peptide-amphiphile (PR_b) was synthesized with a peptide headgroup composed of four building blocks: a spacer; RGDSP, the primary recognition site for alpha5beta1; PHSRN, the synergy binding site; and a linker. The linker was designed to mimic two important criteria: the distance and the hydrophobicity/hydrophilicity between PHSRN and RGD in fibronectin. Human umbilical vein endothelial cells were seeded on different substrates and evaluated in terms of adhesion, spreading, specificity, cytoskeleton organization, focal adhesions, and secretion of extracellular fibronectin. This peptide was shown to perform comparably to fibronectin, indicating that a biomimetic approach can result in the design of novel peptides with therapeutic potential for biomaterial functionalization.     

PEGylated liposomal doxorubicin targeted to α5β1-expressing MDA-MB-231 breast cancer cells

Targeting drugs selectively to cancer cells can potentially benefit cancer patients by avoiding side effects generally associated with several cancer therapies. One of the attractive approaches to direct the drug cargo to specific sites is to incorporate ligands at the surface of the delivery systems. Integrin α(5)β(1) is overexpressed in tumor vasculature and cancer cells, thus making it an attractive target for use in drug delivery. Our group has developed a fibronectin-mimetic peptide, PR_b, which has been shown to bind specifically to integrin α(5)β(1), thereby providing a tool to target α(5)β(1)-expressing cancer cells in vitro as well as in vivo. Our current work focuses on designing modified stealth liposomes (liposomes functionalized with polyethylene glycol, PEG) for combining the benefits associated with PEGylation, as well as imparting specific targeting properties to the liposomes. We have designed PEGylated liposomes that incorporate in their bilayer the fibronectin-mimetic peptide-amphiphile PR_b that can target several cancer cells that overexpress α(5)β(1), including the MDA-MB-231 breast cancer cells used in this study. We have encapsulated doxorubicin inside the liposomes to enhance its therapeutic potential via PEGylation as well as active targeting to the cancer cells. Our results show that PR_b-functionalized stealth liposomes were able to specifically bind to MDA-MB-231 cells, and the binding could be controlled by varying the peptide concentration. The intracellular trafficking of the doxorubicin liposomes was examined, and within minutes after delivery the majority of them were found to be in the early endosomes, whereas after a longer period of time they had accumulated in the late endosomes and lysosomes. The functionalized liposomes were found to be equally cytotoxic as the free doxorubicin, especially at higher doxorubicin concentrations, and provided higher cytotoxicity than the nontargeted and GRGDSP-functionalized stealth liposomes. Thus, the PR_b-functionalized PEGylated nanoparticles examined in this study offer a promising strategy to deliver their therapeutic payload directly to the breast cancer cells, in an efficient and specific manner.     

Surface presentation of bioactive ligands in a nonadhesive background using DOPA-tethered biotinylated poly(ethylene glycol)

We have developed surfaces for the selective presentation of biotinylated peptides and proteins in a background that resists nonspecific protein adsorption; controlled amounts of biotinylated poly(ethylene glycol) (MW 3400 Da; PEG3400) anchored to titanium-dioxide-coated surfaces via an adhesive tri-peptide sequence of L-3,4-dihydroxyphenylalanine (DOPA3-PEG3400-biotin; DPB) were incorporated within a DOPA3-PEG2000 background. Using optical waveguide lightmode spectroscopy, we found that the amounts of sequentially adsorbed NeutrAvidin and singly biotinylated molecules increased proportionally with the amount of DPB in the surface. Biotinylated peptides (MW approximately 2000 Da) were able to fill all three of the remaining avidin-binding sites, while only one molecule of biotinylated PEG5000 or stem cell factor bound to each avidin. The resulting biotin-avidin-biotin linkages were stable for prolonged periods under continuous perfusion, even in the presence of excess free biotin. Hematopoietic M07e cells bound to immobilized peptide ligands for alpha5beta1 (cyclic RGD) and alpha4beta1 (cylic LDV) integrins in a DPB-dose-dependent manner, with near-maximal binding to cylic LDV for surfaces containing 1% DPB. Multiple ligands were adsorbed in a controlled manner by incubating NeutrAvidin with the respective ligands in the desired molar ratio and then adding the resulting complexes to DPB-containing surfaces. Cell adhesion to surfaces containing both cylic LDV and cyclic RGD increased in an additive manner compared to that for the individual ligands. The bioactivity of adsorbed biotinylated stem cell factor was retained, as demonstrated by DPB-dose-dependent M07e cell adhesion and ERK1/2 activation.     

Enhanced integrin mediated signaling and cell cycle progression on fibronectin mimetic peptide amphiphile monolayers

In recent years, a variety of biomimetic constructs have emerged which mimic the bioactive sequences found in the natural extracellular matrix (ECM) proteins such as fibronectin (FN) that promote cell adhesion as well as proliferation on artificially functionalized interfaces. Much interest lies in investigating the ability of the ECM mimetic materials in regulating a number of vital cell functions including differentiation, gene expression, migration, and proliferation. A peptide amphiphile PR_b containing both the cell adhesive GRGDSP and synergistic PHSRN peptide sequences was developed in our group that was shown to support enhanced cell proliferation and ECM FN secretion as compared to GRGDSP and FN functionalized interfaces. In this study, we have investigated the binding affinity of the PR_b peptide ligand with the FN cell surface receptor, the α(5)β(1) integrin. We compared PR_b functionalized surfaces with FN and BSA coated surfaces and GRGDSP functionalized surfaces in terms of promoting intracellular signaling cascades that are essential for enhanced cellular activity. Specifically, we studied the phosphorylation of focal adhesion kinase (FAK) at tyrosine residues Y397 and Y576 and the formation of cyclin D1, both of which are intracellular markers of integrin mediated attachment of cells, signaling pathways, and progression of cell cycle. FAK and cyclin D1 encourage enhanced cell proliferation, differentiation, and gene expression. Our results show that the PR_b peptide ligand has a specific and strong binding affinity for the α(5)β(1) integrin with a dissociation constant of 76.3 ± 6.3 nM. The PR_b peptide ligands supported enhanced FAK phosphorylation activity and increased cyclin D1 formation as compared to the widely used GRGDSP ligand, the native protein FN (positive control), and BSA nonadhesive surfaces (negative control). These results encourage the use of the FN mimetic PR_b peptide in functionalizing biomaterials for potential tissue engineering and therapeutic applications.     

Superhydrophilic zwitterionic polymers stabilize liposomes

Nonionic polyethylene glycol (PEG) as a stealth polymer destabilizes liposomes due to its amphiphilic property. As a result, PEGylated liposomes have to be further stabilized, such as by using a large amount cholesterol. This is a long existing dilemma faced by PEG. In this work, we show that zwitterionic poly(carboxybetaine) (PCB) stabilizes liposomes because of its superhydrophilic nature, thus solving this dilemma. Specifically, PCB-modified liposomes without cholesterol exhibited good retention of hydrophilic drug and long blood circulating characteristics in vivo. To further validate this new PCB chemistry, PCB liposomal doxorubicin without cholesterol was compared with DOXIL for their antitumor therapeutic efficacies.     

Preparation and biological activities of a bivalent poly(ethylene glycol) hybrid containing an active site and its synergistic site of fibronectin

A bivalent poly(ethylene glycol) or PEG hybrid of fibronectin-related peptides was prepared. An active site peptide (RGD) and its synergistic site peptide (PHSRN) of fibronectin were conjugated with an amino acid-type PEG (aaPEG) to form PHSRN-aaPEG-RGD. A moderate spatial array between RGD and PHSRN in fibronectin may be required for synergic activity. The bivalent hybrid exhibited potent cell spreading activity and exhibited potent anti-metastatic activity in a model of experimental metastasis with B16-BL6 cells in mice. PEG may serve as a spacer for maintaining the desired spatial array.     

Antitumor effect of liposomal histone deacetylase inhibitor-lipid conjugates in vitro

Histone deacetylase inhibitor (HDACI), suberoylanilide hydroxamic acid (SAHA), approved by the Food and Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma, is a promising new treatment strategy for various cancers. In this study, we hypothesized that a liposomal formulation of HDACI might efficiently deliver HDACI into tumors. To incorporate HDACI efficiently into the liposomal membrane, we synthesized six HDACI-lipid conjugates, in which polyethylene glycol(2000) (PEG(2000))-lipid or cholesterol (Chol) was linked with a potent hydroxamic acid, HDACI, SAHA or K-182, by cleavable linkers, such as ester, carbamide and disulfide bonds. Liposomal HDACI-lipid conjugates were prepared with distearoylphosphatidylcholine (DSPC) and HDACI-Chol conjugate or with DSPC, Chol and HDACI-PEG-lipid conjugates, and their cytotoxicities were evaluated for human cervix tumor HeLa and mouse colon tumor Colon 26 cells. Among the liposomes, liposomal oleyl-PEG(2000)-SAHA conjugated with SAHA and oleyl-PEG(2000) via a carbamate linker showed higher cytotoxicity via hyperacetylation of histone H3 and induction of caspase 3/7 activity. These results suggested that liposomal HDACI-lipid conjugates may be a potential tool for cancer therapy.     

A reduction-triggered delivery by a liposomal carrier possessing membrane-permeable ligands and a detachable coating

To control the cellular uptake of drugs and genes, we synthesized a liposomal carrier possessing membrane-permeable ligands and a detachable poly(ethylene glycol) (PEG) coating. For the detachable coating, a lipid having a thiolytic cleavable spacer (PEG-S-S-DOPE) was synthesized by the reaction of dioleoylphosphatidylethanolamine (DOPE) with a PEG chain via a disulfide linkage. The liposomes were prepared from a mixture of dipalmitoylphosphatidylcholine (DPPC), DOPE, PEG-S-S-DOPE, and cholesteryl hemisuccinate (CHEMS). The octamer (R8 peptide) of arginine was chosen as the membrane-permeable ligand and covalently immobilized onto the CHEMS portion of the liposome surface (PEG-S-S-R8-liposome). The disulfide bond of the PEG chain was cleaved to display the R8 peptides on the liposome surface by adding a reducing agent such as L-cysteine, and thereby internalization of the liposomes was significantly facilitated. When L-cysteine was added to the mixture of cells and the liposome that incorporated plasmids encoding the enhanced green fluorescence protein (pEGFP), the expression of EGFP was low but could be observed in almost 100% of the cells.     

Effects of poly(ethylene glycol) (PEG) chain length of PEG-lipid on the permeability of liposomal bilayer membranes

The effects of poly(ethylene glycol) (PEG) chain length of PEG-lipid on the membrane characteristics of liposomes were investigated by differential scanning calorimetry (DSC), freeze-fracture electron microscopy (FFEM), fluorescence polarization measurement and permeability measurement using carboxyfluorescein (CF). PEG-liposomes were prepared from mixtures of dipalmitoyl phosphatidylcholine (DPPC) and distearoyl phosphatidylethanolamines with covalently attached PEG molecular weights of 1000, 2000, 3000 and 5000 (DSPE-PEG). DSC and FFEM results showed that the addition of DSPE-PEG to DPPC in the preparation of liposomes caused the lateral phase separation both in the gel and liquid-crystalline states. The fluidity in the hydrocarbon region of liposomal bilayer membranes was not significantly changed by the addition of DSPE-PEG, while that in the interfacial region was markedly increased. From these results, it was anticipated that the CF leakage from PEG-liposomes is accelerated compared with DPPC liposomes. However, CF leakage from liposomes containing DSPE-PEG with a 0.060 mol fraction was depressed compared with regular liposomes, and the leakage decreased with increasing PEG chain length. Furthermore, the CF leakage from liposomes containing DSPE-PEG with a 0.145 mol fraction was slightly increased compared with that of liposomes containing DSPE-PEG with a 0.060 mol fraction. It is suggested that the solute permeability from the PEG-liposomes was affected by not only properties of the liposomal bilayer membranes such as phase transition temperature, phase separation and membrane fluidity, but also the PEG chain of the liposomal surface.     

Surface modification of RGD-liposomes for selective drug delivery to monocytes/neutrophils in brain

In the present study, RGD peptide was coupled with ferulic acid (FA) liposomes for binding to monocytes and neutrophils in peripheral blood for brain targeting in response to leukocyte recruitment. Cholesterol (Ch) was esterified with succinic anhydride to introduce a carboxylic end group (Ch-COOH). Soybean phosphatidylcholine, cholesterol and Ch-COOH were in a molar ratio of 1 : 0.23 : 0.05. FA was loaded into liposomes with 80.2+/-5.2% entrapment efficiency (EE) using a calcium acetate gradient method since it was difficult to load FA by other methods. RGD peptide was a novel compound coupled with Ch-COOH via carbodiimide and N-hydroxysulfosuccinimide. The results of the in vitro flow cytometric study showed that RGD conjugation liposomes (RGD-liposomes) could bind to monocytes/neutrophils efficiently. The rats were subjected to intrastriatal microinjections of 100 microl of human recombinant IL-1beta to produce brain inflammation and subsequently sacrificed after 15, 30, 60 and 120 min of administration of three formulations (FA solution, FA liposome, RGD-coated FA liposome). The body distribution results showed that RGD-liposomes could be directed to the target site, i.e. the brain, by cell selectivity in case of an inflammatory response. For RGD coated liposomes, the concentration of FA in brain was 6-fold higher than that of FA solution and 3-fold higher than that of uncoated liposomes. MTT assay and flow cytometry were used in the pharmacodynamic studies where it was found that FA liposomes exhibited greater antioxidant activity to FA solution on U937 cell.     

Oligomethylene spacer length dependent interaction of synthetic galactolipids incorporated in phospholipid layers with ricin

As models of naturally occurring glycolipids, structurally well-determined amphiphilic compounds were prepared. The synthetic molecules have beta-d-galactopyranosyl or alpha-d-mannopyranosyl and two dodecyl groups as terminal hydrophilic sugar and hydrophobic hydrocarbon moieties, respectively. The two long alkyl chains are connected by 3,5-dioxybenzamide through ether linkages to give a lipid analog purified easily due to its absorbance of ultraviolet light. In the synthetic glycolipids, the glycoside and lipid parts are covalently bound via an oligomethylene spacer. The glycolipids could be easily incorporated into liposomes of l-alpha-phosphatidylcholine. The monoglycosyl moiety of the synthetic glycolipids possessing a hexamethylene spacer was present on the surface of the liposomes and interacted specifically with a lectin to give liposomal assemblies. Such agglutination of these liposomes induced by lectins was determined by analyses of turbidity and particle size based on dynamic light scattering and laser diffraction methods. The other liposomes possessing a shorter ethylene or longer decamethylene linker gave few lectin-induced agglutinates, indicating that these spacers were not effective for the presentation of the galacto-terminal on the liposomal surfaces. Similar spacer-dependent recognition of ricin with a galactolipid-incorporated phospholipid monolayer was confirmed by surface plasmon resonance technique on a substrate.     

Properties of polyethylene glycol supported tetraarylporphyrin in aqueous solution and its interaction with liposomal membranes

5,10,15,20-Tetrakis(4-hydroxyphenyl)porphyrin was functionalized by covalent attachment of poly(ethylene glycol) (PEG) chains of various molecular weights, 350, 2000, and 5000 Da. The properties of PEG-functionalized tetraarylporphyrins in aqueous solution and their interactions with liposomes have been studied. Electronic absorption spectroscopy, dynamic light scattering, atomic force microscopy, and fluorescence quenching were used to monitor aggregation of porphyrin chromophores and behavior of the attached PEG chains in the aqueous solution. The tendency for aggregation of porphyrin chromophores in aqueous solution and the efficiency of fluorescence quenching by KI decrease with increasing length of PEG chain linked to the porphyrin ring. The experimental results indicate that polymer clusters are present in aqueous solution of all pegylated porphyrins. The interactions between the pegylated porphyrins and phosphatidylcholine liposomes in the aqueous solution were studied using the fluorescence methods. The apparent binding constants of porphyrin chromophores to liposomes were determined. The degree of binding was found to be dependent on the molecular weight of the attached polymer.     

Comparison of photodynamic efficacy of tetraarylporphyrin pegylated or encapsulated in liposomes: In vitro studies

Two photosensitizing systems: (1) tetrakis(4-hydroxyphenyl)porphyrin (p-THPP) encapsulated in sterically stabilized liposomes (SSL) and (2) p-THPP functionalized by covalent attachment of poly(ethylene glycol) (p-THPP–PEG₂₀₀₀) were studied in vitro. The dark and photo cytotoxicity of these systems were evaluated on two cell lines: HCT 116, a human colorectal carcinoma cell line, and DU 145, a prostate cancer cell line and compared with these determined for free p-THPP. It was demonstrated that both encapsulation in liposomes as well as attachment of PEG chain result in pronounced reduction of the dark cytotoxicity of the parent porphyrin. The liposomal formulation showed higher than p-THPP–PEG₂₀₀₀ photocytotoxicity towards both cell lines used in the studies.     

Photocontrol of cell adhesion processes: model studies with cyclic azobenzene-RGD peptides

A photoresponsive integrin ligand was synthesized by backbone-cyclization of a heptapeptide containing the integrin binding motif Arg-Gly-Asp (RGD) with 4-(aminomethyl)phenylazobenzoic acid (AMPB). Surface plasmon enhanced fluorescence spectroscopy showed that binding of the azobenzene peptide to alpha(v)beta(3) integrin depends on the photoisomeric state of the peptide chromophore. The higher affinity of the trans isomer could be rationalized by comparing the NMR conformations of the cis and trans isomers with the recently solved X-ray structure of a cyclic RGD-pentapeptide bound to integrin.     

Effects on peptide binding affinity for TNFα by PEGylation and conjugation to hyaluronic acid

Conjugation of cytokine-neutralizing monoclonal antibodies (mAb) to hyaluronic acid (HA) having M_w of 1.6 MDa was previously shown to be an effective strategy for localized delivery to sites of inflammation. Despite the disparity in size of the mAb and HA, the mAb–HA conjugate was found bind tumor necrosis factor-α (TNFα) as strongly as the non-conjugated antibody, suggesting conjugation to this charged polysaccharide can provide an alternative to poly(ethylene glycol) (PEG) conjugation, which has been shown to reduce binding interactions for many proteins. To explore conjugation chemistries more systematically, we report a study on a model peptide inhibitor of tumor necrosis factor-α to investigate the effects of site-specific conjugation to HA and PEG. We compared the binding affinities of a variety of WP9QY peptide–polymer conjugates for TNFα in order to examine the effects of PEG molecular weight as well as the effects of PEG versus functionalized hyaluronic acid (HA) conjugation. The results indicate that the binding affinity of the PEG conjugates decreases in comparing PEG with mass 2 k, 10 k, and 30 k, which was attributed to PEG shrouding of the peptide, while conjugation to a 66 kDa HA chain preserved peptide binding affinity. We attribute this difference to the increased solubility of HA compared to PEG, potentially due to the carboxylic acid functional groups. In addition, the results demonstrate that conjugation to HA via a short PEG linker significantly enhances the association rate k_on, which may reflect an increased peptide accessibility. By balancing both the advantages associated with the PEG conjugates and with the HA conjugates, the HA–PEG2k–WP9QY conjugate was able to improve the binding affinity of the peptide for TNFα by a factor of two. Optimization of polymer chemistry could be used to improve delivery of protein therapeutics for localized and systemic administration.     

Structural Features of Interacting Complementary Liposomes Promoting Formation of Multicompartment Structures

The structural features of complementary liposomes and factors favoring formation of multicompartment systems are investigated. Specifically, liposomal formulations consisting of PEGylated unilamellar liposomes with guanidinium moieties located at the distal end of polyethylene glycol (PEG) chains interact with complementary multilamellar liposomes bearing phosphate moieties. Furthermore, the number of PEG chains attached to the unilamellar interface of the liposomes is enhanced by incorporating PEGylated cholesterol in their bilayer. While molecular recognition of the liposomes is the driving force for initiating multicompartmentalization, it is the enhanced PEGylation at the liposomal interface that synergistically promotes fusion resulting in large and well‐formed multicompartment systems. A mechanism is proposed according to which initial adhesion of the liposomes, followed by reorganization of their membrane lipids, leads to giant bilayer aggregates incorporating large liposomes.     

Artificial ditopic Arg-Gly-Asp (RGD) receptors

Covalent fusion of two artificial recognition motifs for arginine and aspartate resulted in a new class of ditopic RGD receptor molecules, 1-4. The two binding sites for the oppositely charged amino acid residues are linked by either flexible linkers of different length (in 1-3) or a rigid aromatic spacer (in 4). These spacers are shown to be critical for the complexation efficiency of the artificial hosts. If the linkers are too flexible, as in 1-3, an undesired intramolecular self-association occurs within the host and competes with, and thereby weakens, substrate binding. The rigid aromatic linker in 4 prevents any intramolecular self-association and hence efficient RGD binding is observed, even in buffered water (association constant of K(a) approximately 3000 m(-1)). A further increase in hydrophobic contacts, as in host 16, can complement the specific Coulomb attractions, thereby leading to an even more stable complex (Ka=5000 m(-1)). The recognition events have been studied with NMR spectroscopy, UV/Vis spectroscopy, and fluorescence titrations.     

Effects of poly(ethylene glycol) (PEG) concentration on the permeability of PEG-grafted liposomes

Poly(ethylene glycol)-grafted liposomes (PEG-liposomes) were prepared from dipalmitoylphosphatidylcholine (DPPC) with various amounts of distearoyl-N-monomethoxy poly(ethylene glycol)-succinyl-phosphatidylethanolamines (DSPE-PEG) with PEG molecular weights of 1000, 2000, 3000 and 5000. The effects of DSPE-PEG concentration on the permeability of PEG-liposomes were investigated using carboxyfluorescein (CF). In the gel state, the CF leakage from PEG-liposomes was decreased with increasing mole fractions of DSPE-PEG for all PEG molecular weights. In the liquid-crystalline state, the CF leakage from PEG-liposomes containing DSPE-PEG1000 gradually increased with increasing mole fractions of DSPE-PEG, while that of PEG-liposomes whose molecular weight in PEG units was above 2000 rapidly decreased by the addition of DSPE-PEG. Furthermore, no effect of PEG molecular weight on CF leakage was observed. The relationship between the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) (or 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH)) and the mole fraction of DSPE-PEG for PEG-liposomes was also investigated. No significant changes in fluorescence polarization of DPH for liposomal bilayer membranes was observed in the gel and liquid-crystalline states due to the addition of DSPE-PEG, while that of TMA-DPH was decreased compared with that of liposomes without DSPE-PEG in both states.     

Adhesive peptides conjugated PAMAM dendrimer as a coating polymeric material enhancing cell responses

This present work aims to functionalize poly(amidoamine) (PAMAM) dendrimers with various reported adhesive peptides, including Arg-Gly-Asp (RGD), Tyr-Ile-Gly-Ser-Arg (YIGSR), and Ile-Lys-Val-Ala-Val (IKVAV) for enhancing cell responses. The RGD, YIGSR, or IKVAV functionalized PAMAM coated substrate could promote cell adhesion of bone marrow mesenchymal stem cells (BMSCs) within 1 h after incubation. The neurite differentiation and proliferation of pheochromocytoma (PC12) cells were also significantly enhanced after culturing on the peptide functionalized PAMAM dendrimers for two and four days. This peptide functionalized PAMAM dendrimers are considered as the potential candidates for various tissue engineering applications.